Electrophoresis, in general, is the phenomenon of the migration of charged particles or ions in a liquid carrier medium under the influence of an electric field. This phenomenon can be used to separate small particles which, by reasons of different surface chemical properties, exhibit different concentrations of surface charge in the given medium. Under the influence of the electrical field, the electrophoretic mobilities of the various classes of charged particles in the carrier medium will be different. A sample introduced at some point into the sheet of liquid carrier medium (buffer) diffuses slowly in a narrow band in the absence of a potential gradient; however, when the potential gradient is applied to the sheet of buffer, the sample particles are separated under the influence of the electrical field into various particle groups or components depending upon the electrophoretic mobility of the respective particles, the strength of the field, and the length of time that the particles remain in the field. Particles of similar mobility are concentrated in distinctive zones or bands at defined distances from the point of sample introduction (origin).
Blotting or transfer of electrophoretically resolved material, such as DNA, RNA, and protein, has become a standard procedure when sensitive and specific detection of biologically interesting macromolecules is required.
Electroblotting offers significant advantages over capillary blotting in that the electroblotting procedure is much quicker. Capillary transfer and electroblotting both require that the gel be placed in contact with the paper or other membrane to which the proteins or nucleic acids or other materials will be transferred. The difference between the methods is the transfer driving force. In capillary transfer the driving force is the absorptive potential of the filter paper, or other material. The transfer material, e.g., nitrocellulose or nylon, is placed between the gel and the absorptive paper. In electroblotting, however, as currently practiced the gel and transfer material are vertically suspended in a buffer tank between two electrodes. The protein or nucleic acids are thus driven out of the gel onto the transfer material using electrical potential. For example, a typical system involves placing a nylon membrane against a gelatin sheet, submerging the gel-nylon assembly vertically into a buffer solution, then applying an electric potential transversely across the assembly using the buffer solution as the conducting medium. This system typically uses two platinum wire electrodes, one on each side of a gel-nylon combination, and establishes a voltage gradient in the buffer solution. The electrodes are laid out in grid fashion and spaced at a distance from the gel and nylon to obtain a reasonably uniform electric field using the least amount of platinum.
The blotting procedure offers significant advantages. Firstly, molecules in the matrix of a gel are relatively inaccessible to probes such as antibodies. Transfer to the surface of a membrane allows analyses that are difficult or impossible in the gel. Also, since the transferred molecules are located at or near the surface of the membrane, analysis time is substantially reduced. In addition, the membranes are relatively strong and easy to handle in contrast to the gels which are easily torn. Moreover, the transferred molecules are bound to the membrane so that there is no loss of resolution while biological activity is usually retained. Thus, storage of the membrane prior to use is usually feasible.
As mentioned hereinabove, in electroblotting as currently practiced the gel and transfer material are vertically suspended in a buffer tank between two electrodes. A number of disadvantages are associated with vertical electroblotting. For example, battery jar-type tanks limit the size of the gel blot possible while blotting tanks for blotting large electrophoresis gels require excessive amounts of buffer and are very expensive to construct. In addition, holders for vertical blotting systems must be very strong to maintain close proximity of the gel and blotting membrane. This requires large support systems which tend to block the electrical charge thereby leading to blurred blots. The size of the support system also limits the size of the gel blot possible. Finally, soft agarose gels, such as those required for genome identification, cannot be blotted in a vertical electroblotting system due to the slippage, sliding and collapsing of the gel in the support holder.
Attempts at horizontal blots, which would overcome some of the problems noted above with regard to vertical blotting systems, have been largely unsuccessful. During electroblotting in horizontal blotting systems oxygen and hydrogen bubbles are created at the cathode and anode respectively during electrolysis. The bubbles given off at the electrode beneath the horizontally disposed gel membrane accumulate on the lower surface of the membrane thereby partially blocking the electrical charges which results in uneven blotting.
Naturally, it would be highly desirable to provide a horizontal electroblotting system suitable for use with soft agarose gels and the like wherein bubbles which are created at the electrodes during electrolysis are dispersed in such a manner as to prohibit accumulation of bubbles on the undersurface of the horizontally disposed gel membrane.
Accordingly, it is the principal object of the present invention to provide a device for the horizontal electroblotting of electrophoretically transferred material.
It is a further object of the present invention to provide a device as aforesaid which is particularly suitable for use with soft gels such as agarose gels and the like.
It is a particular object of the present invention to provide a device as aforesaid which prevents accumulation of bubbles on the undersurface of the horizontally disposed gel membrane.
It is a still further object of the present invention to provide a device as aforesaid which is of simple construction, economic to manufacture and easily used in electroblotting.
Further objects and advantages of the present invention will appear hereinbelow.